Electrolytic preparation of a dihydrostreptomycin sulphate



United States PatenfO ELECTROLYTIC PREPARATIONIOF A DIHYDRO- STREPTOM'YCIN SULPHATE Morris A.- Dolliver, Stelton, vand-Serge Semenofi; New Brunswick, N. J., assignors,:by mesne-assignments, to Olin Mathieson Chemical Corporation, a corporation of Virginia No Drawing; ApplicationDecember-9,1949? Serial'No; 152,21! f 1 Claim;..-(Cl. 204 75):

This invention is concerned with the production of dihydrostreptomycins. These compounds have been obtained heretofoiewby :treatment f the corresponding streptomycins withihydrogen-in the presence of=a-noble metal catalyst, a relatively -expensiveproceSs-in that it necessitates recovery' of "the: catalystand replacement: of catalyst inevitably lostin: the: recovery. [-Tliei terms streptomycins anda streptomycin employedherein have reference tozthe recognized igenus composed of streptomycin, "mannosidostreptomycin and"streptomycin complex; and the terms F dihydrostreptomycins and a dihydrostreptomycim?havereference to the' corresponding genus comp'osed of. dihydro .deriva'tivesofi streptomycin, etc." Also,.all of ithesequoted'terms; when employed unmodified. hereinafter; have reference to these in their. usual free-base"and/owwatei soluble-acidaaddi tion forms] I It has been. found: that =dihydrostreptomycinsmay'ad vantageously be produced by electrolyticreduction," i. e., bya process essentially-comprising charging-angelec trolytic cell, having anode and cathode compartmentsseparated .by a semi-permeablediaphragm,v witha nonalkaline, electric-current-conducting aqueous solution of the corresponding streptomycin as the'cathol'yte and an aqueous acid as the-anolyte', passing an electric. current between the anode and cathode. in the-respective com partrnents until thestreptomycin is substantiallyi com pletelyreduced, and recovering the dihydro'streptomycin' from the catholyte. v

Inasmuch. as the process of'this invention may'be=car1- ried out in electrolytic-reduction apparatus generallyi: e., does not require any special type), -detailed-descrip'-- tion and drawing of such apparatus-is unnecessary. A simple electrolytic-reduction'-cell for laboratory use is described,-forexample, on pages 10-12 of-Organie-Sym theses, vol. 21 published: in 1941 -by-John Wiley & Sons, Inc.,. New York; it consists ofa glass beaker eontaining a smaller. volume porous cup, 'a' mechanical unrem rside the porous cup, 'and sheet lead-electrodes one'being' within the porous cup), the electrodes rbeing ina directcurrent circuit having an.ammeter and variable resistance,

' and the beakerbeing in a co'oling bath iThecatholyte is placed in the cathode chamber between th'epe'rmeable diaphragm provided by the-porouscup and -thezbaker wall, andthe anolyte within the-porous cup'g and r'ed'ua tion is effected .while sti-rring' and coolingwuntil 'substantially complete, asevidcnced for exampleby the-increasedevolution of hydrogen. i

The semi-permeable diaphragm of electrolytic-reduction apparatusis a diaphragmwhi'clrpermits hydro gen ions to pass from: the anode compartment -into:- the cathode compartment and permits the passage 'of anions (e. g.,. sulfate or'chloride) from the-cathode compartment to the anode compartment; but is sutficiently impermeable to' prevent: diffusion of the -catholyte-com ponent to be reduced intoithe anode compartment. Various materials are" suitable forbuse -as se rni-permeable dia' phragms for electrolytic"reduction?and may besous'ed for the purposes-of this inventiong intercalia,.-unglazed porcelain, thin. cork-pine wood, filtros, ands notably Alundum. I

The electrodes-for the purposesofil thisinvention/may be of any of the materials commonly used for electro lytic reduction, provided thatnthencathode-is :05 a material non-reactive with the streptomycins and I :dihydrostreptomycins. Thus, the anode: maybe of .lead lead coated with lead dioxide,-platinum,=. carbon,.graphiteptin, zinc, copper, nickel, :mercury,- amalgamated-zinc, and amalgamated lead, inter- 'alia; andl thescathode -may be of carbon, lead, amalgamated lead, mercur-y,. anoble-metal (e. g., platinum), and .lead alloyedewithl.otherqrnetals (e. g., a solder containing. 40% tin,.or leadacontaining about 1-15% antimony);-inter. alia. Preferably, ,the cathode is metallic, notably; essentially leadu Preferably also, the electrodeslespecially theycathodel-are in ex tended surface. form, e. g., inv the form-ofacoilsorsheets, or spongy;,and the anode. and. cathode have substantially the same surface area.

The anolytefor the. purposes ofthis inventionimay be of any of the. compositionscommonly.usednforuelectro lytic reduction, e. g.,- aqueous solutions.of various-xcom centrations of strong inorganic-acids;-such-,aswulfuric; hydrochloric, or phosphoricl acid...,..When electric vcur= rent .is passed through the cell, .sulfate.ions (for. example)' are discharged. at the anode,-reacting with thmwa'ter to form oxygen and sulfuric acid; Preferably,,ethe ano-j lyte is sulfuric acidina concentration-o about. 0.5%:"to about 80%, notably a concentration of about 33%;.--

The catholyte for the purposes of this invention is a non-alkaline, electric-current-conducting aqueous solutionof the corresponding streptomy'cirnil a; a solution; in which the streptomycin 'exist s at least in partin the form of its water-solubleacid ad'dition :saltj'or-a' solution in which there is present in addition-to streptomycinsuifi cient water-soluble acid to make the solutionfnon' all'taline and electric-current-conducting;- Pr'eferabl'yfi'the catholyte is anaqueous .solution'of the' hydrochlb'rideor sulfate ofthe streptomycin. When electric"current=iis passed through-the cell; hydrogen" ions migrate to the cathode and are discharged asnascent-"hydrdgen;which reacts with the streptomycin in thecatholyte'tofoirn the corresponding dihydrostreptomycin. Inasmuch as' the catholyte-tends to become-alkaline during-theelectro1ysis; dilute acid is added during electrolysis to-maintain the catholyte' nonalkaline', preferably at' a pH of about 4 to about 7. j v

The direct current employed-may be at'various voltages (e. g., 6 volts or 110 volts), the desired amperage .being'ob' tained by suitable resistances. Electrolysis may beffted at various amperages (e'. g.,.tliampere and amperes), and at various temperatures below that at .which the streptomycin decomposes in the catholyt'e (e;' g.,,btween about C." and' about 40"-",C.).. The higher amperage require more efiicient cooling of the electrolytic reduction cell. The temperature, voltage; amperage and current density are not critical factors in the process of this invention.

.By the process of this invention, dihydrostreptomycin, 'dihydromannosidostreptomycin, and dihydrostreptomyci-n. complex'may be etliciently'produced from the correspond.- ing'streptomycins (streptomycin or streptomycinJA,- man-. nosidostreptomycin or streptomycin B, and' streptomycin complex, respectively). The concentration of the streptomycin. in the catholyte is :not' critical, concentrations of streptomycin sulfate of about10% 'and ab'out- -foi example, being feasible.

The process of this invention is particularly advantage-t:

sulfuric'acid to the catholyte during electrolysis, one obtains the corresponding dihydrostreptomycin sulfate substantially free from the corresponding hydrochloride salt and yielding only a faint test for chloride. Inasmuch as crystalline streptomycins are most conveniently obtained as the chlorides, and the dihydrostreptomycins are more desirable physiologically in the form of their sulfates, the process of this invention may be regarded as an advantageous process of converting the streptomycin chloride to the desirable sulfate of the dihydro derivative. By employing the desired acid as the acid added to the catholyte during electrolysis, any desired-acid salt of the streptomycin may be obtained by the process of this invention.

The dihydrostreptomycin formed may be conveniently recovered as its acid-addition salt by removing the catholyte from the cell and freeze-drying the solution; and when the streptomycin employed is of therapeuticallyutilizable purity, the dihydrostreptomycin obtained is suitable for therapeutic use without further purification. Further purification of a dihydrostreptornycin obtained by the process of this invention may be effected in the same manner as the purification of a similarly impure dihydrostreptomycin obtained by catalytic hydrogenation.

The process of this invention may of course be carried out in apparatus designed for large scale operation. Thus, the apparatus may include a separate cooling chamber and/or a pH and concentration-adjusting chamber for the catholyte, and means for circulating the catholyte from the cathode chamber to said chamber or chambers and back; and one may employ a number of electrolyticreduction cells in series, these and other expedients for large scale production being conventional.

The following examples are illustrative of the invention:

Example 1 The electrolytic reduction is carried out in a cell divided into anode and cathode compartments by means of a porous alundum diaphragm, with lead electrodes made of strips of sheet lead containing about 2% antimony, the cathode being helical in shape. The cathode compartment is charged with a 25% aqueous solution of substantially pure streptomycin hydrochloride (810 units/ mg.) as the catholyte, and the anode compartment with 0.5% aqueous sulfuric acid as the anolyte. Direct current from a 6-volt D. C. source is applied to the cell at a current density of 2 amperes/sq. cm., while maintaining the catholyte at pH 7 by dropwise addition of dilute sulfuric acid thereto and the temperature at about 20 to 30 C. by cooling. Reduction to the extent of 99.75% is obtained in six hours. The catholyte is then freeze-dried, yielding dihydrostreptomycin sulfate having a potency of about 756 units/mg. in a yield of about 74%. The product gives only a faint test for chloride ion on treatment with silver nitrate.

Example 2 Example 3 When using carbon (instead of lead) electrodes in Example l, and reducing for 15 hours at a current density of .03 ampere/sq. cm., reduction to the extent of about 58% is obtained.

Example 4 When using platinum (instead of lead) electrodes in Examplel, reduction to the extent of about 72% is ob- F tained in 4 hours at a current density of .01 ampere/sq. cm.

I Example 5 Under the same conditions as in Example except that dilute hydrochloric acid (instead of sulfuric acid) is added to the catholyte to maintain it at pH 7,- dihydrostreptomycin hydrochloride of comparable potency is obtained in substantially the same yield.

Example 6 Under the same conditions as in Example 1, but with a 25% aqueous solution of substantially pure dihydrostreptomycin hydrochloride as the catholyte, substantially pure dihydrostreptomycin sulfate giving only a faint test for chloride ion is obtained.

Example 7 Under the same conditions as in Example 1, but with a 25% aqueous solution of substantially pure mannosidostreptomycin hydrochloride as the catholyte, substantially pure dihydromannosidostreptomycin sulfate is obtained in high yield.

Example 8 (a) A primary streptomycin-containing liquid (obtained, for example, by culturing Streptomyces griseus to produce streptomycin, acidifying the culture, separating the solids from the culture liquid, and neutralizing the separated liquidas described in Rake, Koerber, and Donovick Patent 2,461,922, dated February 15, 1949) is treated with activated charcoal; and the streptomycin is eluted from the charcoal with dilute hydrochloric acid at about C.

(b) The streptomycin-hydrochloride-containing eluate thus obtained, having. a potency of 340 units/mL, is adjusted to pH 6.5 with 2% sodium hydroxide solution, and electrolytically reduced as described in Example 1, adding hydrochloric acid (instead of sulfuric acid) to the catholyte to maintain pH 7, until reduction is complete; and the reduced catholyte is collected.

(c) 10 liters of reduced catholyte obtained as described in (b) is mixed with 36 m1. of a 25% aqueous solution of [e. g., Tergitol Penetrant 7]; and the precipitate formed (a salt-type combination of the dihydrostreptomycin and the surface-active agent) is filtered off and dried. The salt-type combination is then dissolved in ml. methanol, and the impurities filtered off; the filtrate is diluted with 300 ml. water; 60 g. of a neutral anion-exchange resin [e. g., Amberlite IR-4B; cf. U. S. Patent 2,402,384, dated June 18, 1946] is added; the mixture is heated to 50 C.; and concentrated hydrochloric acid is added over a period of a half hour at such rate as to maintain the pH between 3.0 and 3.5. The pH is then allowed to rise to 5.0-5.5, and the resin filtered off and washed with water. The combined filtrate and wash is then vacuum-distilled to remove the methanol, and the residual solution in freeze-dried, yielding 4.6 g. of the hydrochloride of the dihydrostreptomycin, having a potency of 615 units/mg. (yield from eluate, 83.5%).

The invention may be variously otherwise embodied within the scope of the appended claim.

We claim:

The process of converting a streptomycin hydrochloride into a dihydrostreptomycin sulfate, which essentially comprises charging an electrolytic cell, having anode and cathode compartments separated by a semi-permeable diaphragm, with a nonalkaline, electric current-conducting aqueous solution of said streptomycin hydrochloride as the catholyte and an aqueous solution of a strong inorganic acid as the anolyte, the acid being substantially non-reactive with the anode, and the cathode being substantially non-reactive with the components of the catholyte, passing an electric current between the anode and the cathode in the respective compartments while adding sulfuric acid to the catholyte until said hydrochloride salt is substantially completely converted to a sulfate salt, the electrolysis being effected at a temper- References Cited in the file of this patent UNITED STATES PATENTS 2,300,218 Hales Oct. 27, 1942 2,457,933 Spiegelberg Ian. 4, 1949 2,498,574 Peck Feb. 21, 1950 2,590,141 Wolf Mar. 25, 1952 6 Levy Dec. 22, 1953 OTHER REFERENCES Journal American Chemical Society, vol. 68 (July 1946), pp. 1390-91.

Bartz et al., Journal American Chemical Society, vol. 68 (November 1946), pp. 2163-66.

Fried et al., Journal American Chemical Society, vol. 69 (January 1947), PP. 79-86.

Solomons et al., Science, vol. 109 (May 1949), pp. 515-16.

Waksman, Streptomycin (1949), pp. 47-49, 69-70. 

